Saturday, May 10, 2025
5/10/2025
The miR-183/96/182 Cluster in Pseudomonas aeruginosa-induced Keratitis - Pseudomonas aeruginosa (PA) keratitis is one of the most rapidly developing and destructive diseases of the cornea and a global cause of visual impairment and blindness. Emergence of antibiotic-resistant strains poses additional challenges for effective disease management. Development of alternative treatment is urgent. In this regard, microRNAs (miRNAs) are small, non-coding RNAs and important regulators of gene expression. miRNAs play critical roles in human diseases and are viable therapeutic targets. However, the roles of miRNAs in PA keratitis remain largely unexplored. Our long-term objectives are to uncover the molecular mechanisms of miRNAs in ocular infectious diseases, identify novel miRNA-based therapeutic targets and develop alternative treatment of these diseases. The proposed research will directly address this knowledge gap. It is built upon our recently published and strong preliminary data showing that application of anti-miRs targeting the miR-183/96/182 cluster (referred to as miR-183C from here on) and knockout of miR-183C in mice decreases corneal nerve density and neuropeptide production, while reducing the severity of PA keratitis; miR- 183C targets key genes regulating corneal sensory innervation, e.g., Nrp1, bacterium-induced sensory-neuron activation and neuropeptide production, e.g., Toll-Like Receptor (TLR)4, Formyl Protein Receptor (Fpr)1 and substance P (sP) precursor gene Tac1. These data lead us to the overarching hypothesis that, in addition to innate immunity, miR-183C modulates PA keratitis through its regulation of corneal sensory nerve function and neuroimmune interaction by targeting key genes involved in sensory innervation, PA-induced sensory-neuron activation and pro-inflammatory neuropeptide production. Three Aims are proposed in this application to test this hypothesis. In Aim 1, fluorescein amidites (FAM)-labeled anti-miR-183C will be applied to the cornea of wild-type (WT) mice to test the hypothesis that anti-miR-183C treatment upregulates Tac1, TLR4, Fpr1 and Nrp1 in corneal sensory nerves, resulting in increased pro-inflammatory neuropeptides (e.g., sP) and an early immune/inflammatory response (<24 hours post-infection) and an accelerated sensory-nerve reduction and decreased pro-inflammatory neuropeptides and immune/inflammatory response in a later stage during disease resolution. Aim 2 will test the hypothesis using a sensory neuron-specific miR-183C conditional knockout mouse model. Aim 3 will test the hypothesis that knockdown or knockout of miR-183C in vitro/ex vivo in both human and mouse sensory neurons parallels the in vivo data in that it will upregulate Tac1, TLR4 and Fpr1 to enhance PA-induced secretion of pro-inflammatory neuropeptides by sensory neurons, and increase Nrp1 expression to inhibit neurite growth. A genome-wide identification of miR-183C target genes in both human and mouse trigeminal ganglion sensory neurons will also be conducted by RNA seq. This study will provide novel biological insights into molecular bases of miR-183C's regulation of PA keratitis and other ocular infectious diseases. It will uncover mechanisms and reveal new targets for anti-miR therapy.
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